[116] Sodium and potassium-stimulated ATPase

TLDR: The method is described that separates phosphomolybdate from ATP by extraction into butyl acetate and the general principle of purification procedure is to obtain a microsomal fraction by differential centrifugation of a sucrose homogenate and to form dispersed and activated particles with a detergent, urea, or a concentrated iodide solution.

Abstract: Publisher Summary The ATPase activity which requires Na+, K+, and Mg++ together and is inhibited by cardiac glycosides is a part of the enzyme system for the stoichiometric transport of Na+ outward and K+ inward across cell membranes. It is widely distributed in animal tissues and species. Organs, which transport Na+ and K+ to energize electrical activity or secretion, show much activity. The chapter examines the preparation, purification, and properties of sodium and potassium-stimulated ATPase. From the rate of release of inorganic phosphate from ATP in the presence of Na+, K++, and Mg++ is subtracted the rate of release in the presence of Mg++ and a cardiac glycoside inhibitor, such as ouabain. Released inorganic phosphate may be measured simply by adding 2.5 ml of 0.48 M HC104, mixing, filtering, and taking a 2.0-ml aliquot for assay. The method is described that separates phosphomolybdate from ATP by extraction into butyl acetate. The general principle of purification procedure described in the chapter is to obtain a microsomal fraction by differential centrifugation of a sucrose homogenate and to form dispersed and activated particles with a detergent, urea, or a concentrated iodide solution. Aging, freezing and thawing, and mild detergents may improve the activity or sensitivity of fresh homogenates or microsomal fractions. The Km for ATP is about 0.3 mM with Mg++ in slight excess. As the amount of Mg++ is reduced with maximal ATP, the activity decreases but the sensitivity increases. The Km for Na+ is about 1.5 mM and for K+ about 0.4 mM. The apparent affinity of the Na+-site for K+ is about 7-fold less than for Na+ whereas the apparent affinity of the K+-site for Na+ is about 160-fold less than for K+.